Brain-targeted intranasal delivery of protein-based gene therapy for treatment of ischemic stroke

Gene therapy using a protein-based CRISPR system in the brain has practical limitations due to current delivery systems, especially in the presence of arterial occlusion. To overcome these obstacles and improve stability, we designed a system for intranasal administration of gene therapy for the treatment of ischemic stroke. Methods: Nanoparticles containing the protein-based CRISPR/dCas9 system targeting Sirt1 were delivered intranasally to the brain in a mouse model of ischemic stroke. The CRISPR/dCas9 system was encapsulated with calcium phosphate (CaP) nanoparticles to prevent them from being degraded. They were then conjugated with β-hydroxybutyrates (bHb) to target monocarboxylic acid transporter 1 (MCT1) in nasal epithelial cells to facilitate their transfer into the brain. Results: Human nasal epithelial cells were shown to uptake and transfer nanoparticles to human brain endothelial cells with high efficiency in vitro. The intranasal administration of the dCas9/CaP/PEI-PEG-bHb nanoparticles in mice effectively upregulated the target gene, Sirt1, in the brain, decreased cerebral edema and increased survival after permanent middle cerebral artery occlusion. Additionally, we observed no significant in vivo toxicity associated with intranasal administration of the nanoparticles, highlighting the safety of this approach. Conclusion: This study demonstrates that the proposed protein-based CRISPR-dCas9 system targeting neuroprotective genes in general, and SIRT1 in particular, can be a potential novel therapy for acute ischemic stroke.


Figure S1 .
Figure S1.Screening of sgRNAs that guide dCas9-VP64 to activate SIRT1 promoter and preparation of dCas9-VP64 protein.A Designed sgRNAs with different sequences.B SDS-PAGE analysis of dCas9-VP64 proteins.C mRNA expression levels of SIRT1 after the delivery of sgRNA 1, 2 and 3 with dCas9-VP64 proteins in HBECs.Data is expressed as fold change relative to the control after normalization to GAPDH.Error bars indicate mean ± S.D. (n = 3, *P < 0.05 versus control).

Figure S2 .
Figure S2.The encapsulation efficiency of the dCas9-VP64 proteins by western blot.The dCas9-VP64 proteins were obtained from the supernatant after centrifugation under different synthesis conditions of the nanoparticles.Sample loading was 20 μl per well.

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Figure S3.A The FE-SEM image and SEM-energy dispersive X-ray spectroscopy (EDX) analysis of the CaP nanoparticles.B The electron diffraction pattern of the dCas9/CaP/PEI-PEG-bHbs.

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Figure S4.A The stability of the dCas9/CaP/PEI-PEG-bHbs was measured by dynamic light scattering (DLS).B The release pattern of free dCas9 proteins from the nanoparticles was evaluated by western blot.The dCas9 proteins were obtained from the supernatant at various time points, including 0, 1, 3, 5, 10, and 30 days after synthesis.

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Figure S5.A Protein expression levels of monocarboxylate transporter 1 (MCT1) in various cell lines was confirmed by western blot.B After treating the dCas9/CaP/PEI-PEG-bHb in various cell lines, the cells were stained with anti-dCas9 (green), and the nuclei were stained with DAPI (blue).

Figure S6 .
Figure S6.Uptake mechanism of the particles in HNEpCs.dCas9 protein was identified under treatment with various metabolic inhibitors (MCT1 inhibitor, genistein, chlorpromazine, and cytochalasin B) via confocal laser scanning microscopy (CLSM).The nuclei were stained with DAPI (blue) and cells were stained with anti-dCas9 (green).

Figure S7 .
Figure S7.The gene editing capability of the dCas9/CaP/PEI-PEG-bHb in vitro.mRNA and protein expression levels of SIRT1 in HBECs after transferring the particles from HNEpCs treated with various nanoparticles.Data is expressed as fold change relative to the control group after normalization to GAPDH.Error bars indicate mean ± S.D. (n = 3, **P < 0.01 versus control).

Figure S8 .
Figure S8.Schematic illustration of intranasal administration of the nanoparticles in mice.

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Figure S9.A Representative brain tissues stained for dCas9-VP64 proteins.B mRNA and protein expression levels of Sirt1 in the brain after intranasal administration of the various nanoparticles.Data is expressed as fold change relative to the control group after normalization to Gapdh.Error bars indicate mean ± S.D. (n = 10 mice per group, *P < 0.05 versus control).

Figure S10 .
Figure S10.Establishment of a permanent middle cerebral artery occlusion (pMCAO) model for studying ischemic stroke.A Procedure for pMCAO surgery.The CCA, ICA, and ECA are exposed, and a silicone filament is inserted into the CCA and ICA until it reaches the MCA (see the Materials and Methods for details).Figure created with BioRender.com.B Representative photographs of TTC stained brain.The white areas represent the infarcted areas in pMCAO.C mRNA expression levels of Sirt1 in the ischemic brain at 1, 3, and 6 hours after pMCAO.Data is expressed as fold change relative to the sham group after normalization to Gapdh.Error bars indicate mean ± S.D. (n = 3) (n = 10 mice per group, *P < 0.05, *** P < 0.001 versus sham).Abbreviations: CCA, common carotid artery; ICA, internal carotid artery; ECA, external carotid artery; MCA, middle cerebral artery; TTC, 2,3,5triphenyltetrazolium chloride.
Figure S10.Establishment of a permanent middle cerebral artery occlusion (pMCAO) model for studying ischemic stroke.A Procedure for pMCAO surgery.The CCA, ICA, and ECA are exposed, and a silicone filament is inserted into the CCA and ICA until it reaches the MCA (see the Materials and Methods for details).Figure created with BioRender.com.B Representative photographs of TTC stained brain.The white areas represent the infarcted areas in pMCAO.C mRNA expression levels of Sirt1 in the ischemic brain at 1, 3, and 6 hours after pMCAO.Data is expressed as fold change relative to the sham group after normalization to Gapdh.Error bars indicate mean ± S.D. (n = 3) (n = 10 mice per group, *P < 0.05, *** P < 0.001 versus sham).Abbreviations: CCA, common carotid artery; ICA, internal carotid artery; ECA, external carotid artery; MCA, middle cerebral artery; TTC, 2,3,5triphenyltetrazolium chloride.

Figure S11 .
Figure S11.mRNA expression levels of Bcl2, Bax, Mmm9 and Parp in the ischemic brain without nanoparticles.All data are expressed as fold change relative to the sham group after normalization to Gapdh.Error bars indicate mean ± S.D. (n = 10 mice per group, *P < 0.05, *** P < 0.001 versus sham).

Figure S12 .
Figure S12.mRNA expression levels of Bcl2, Bax, Mmp9 and Parp in the ischemic brain 6 h after pMCAO with the various prepared nanoparticles compared to sham surgery.All data are expressed as fold change relative to the sham group after normalization to Gapdh.Error bars indicate mean ± S.D. (n = 10 mice per group, *P < 0.05, **P < 0.01, *** P < 0.001 versus sham).

Figure S13 .
Figure S13.Assessment the effect of the nanoparticles on brain swelling 12 hours after pMCAO.Quantitative analysis of brain water content in the ischemic brain after intranasal delivery of the nanoparticles 3 hours after pMCAO.Error bars indicate mean ± S.D. (n = 3 mice per group, *P < 0.05 versus sham, # P < 0.05, ## P < 0.01 versus pMCAO).(BWC, brain water content).

Figure S14 .
Figure S14.Distribution of the nanoparticles and toxicity in vivo.A mRNA and protein expression levels of Sirt1 in various organs after intranasal administration of the various nanoparticles.B mRNA expression levels of Sirt1 in the brain and lung at 7 days after intranasal delivery of the dCas9/CaP/PEI-PEG-bHb.All data are expressed as fold change relative to the control group after normalization to Gapdh.Error bars indicate mean ± S.D. (n = 10 mice per group).Abbreviations: Empty/CaP/PEI-PEG-bHb, CaP/PEI-PEG-bHb particle that does not contain the dCas9-VP64 protein with sgRNA.

Figure S16 .
Figure S16.H&E staining of various organs from mice not treated and treated with dCas9/CaP/PEI-PEG-bHb.